Effects Of Protein Synthesis Inhibitors Research Articles

P2-226 - Genetic factors influence the effect of protein synthesis inhibition on cocaine-kindled seizures

P2-226 - Genetic factors influence the effect of protein synthesis inhibition on cocaine-kindled seizures

Effects of Protein Synthesis Inhibitors on Uptake of Aluminium in Aluminium-Resistant and Aluminium-Sensitive Cultivars of Wheat

Summary The effects of two inhibitors of protein synthesis on the kinetics of Al uptake by excised roots of Al-resistant cultivars (Atlas 66 and PT 741) and Al-sensitive cultivars (Neepawa and Scout 66) of wheat were investigated. Treatment of intact roots of the Al-sensitive cultivar, Neepawa, with cycloheximide (0 to 5 mM) showed that this inhibitor was more effective in reducing incorporation of 35 S into microsomal membrane proteins than into total proteins. Cycloheximide at 0.25 mM reduced incorporation of 35 S into microsomal membrane proteins by 68 %, while a 21 % reduction into total proteins was observed. Cycloheximide at 1.0 mM was sufficient to induce maximal inhibition of 35 S incorporation into both microsomal membrane (72 %) and total (40 %) proteins. At this concentration, cycloheximide caused quantitative differences in short-term (3 h) Al uptake between Al-resistant and Al-sensitive genotypes. A significant decrease in rates of Al uptake was observed in both Al-sensitive cultivars (24 and 29 %), while no significant effect was observed in the Al-resistant cultivars. These results are in contrast to previous results from long-term experiments (6 and 12 h), where cycloheximide stimulated Al uptake in an Al-resistant cultivar, Atlas 66 (Aniol, 1984; Rincon and Gonzales, 1992). In experiments where Al uptake was measured after a 4-h pretreatment with 1.0 mM cycloheximide (7 h total exposure), a stimulation of uptake (45 %) was observed in the Al-sensitive cultivar, Neepawa. Thus, time of exposure is an important experimental variable that can account for contradicting results in the literature. In both resistant and sensitive cultivars, treatment with chloramphenicol, an inhibitor of prokaryotic protein synthesis, had no effect on the kinetics of Al uptake. Posible mechanisms whereby protein synthesis might affect Al uptake in Al-resistant and Al-sensitive genotypes are discused.

Effects of protein synthesis inhibitor and antimicrotubular agent on transepithelial movement of 3H-androgens in the rat caput epididymis.

The effects of protein synthesis inhibition and disassembly of microtubules in the epididymal epithelia on proluminal movement of 3H-androgens were investigated by using in vivo microperifusion of 3H-testosterone and subsequent micropuncture to obtain peritubular and intraluminal fluids of caput epididymal tubules. Cycloheximide (100 micrograms/ml) was used as protein synthesis inhibitor. Nocodazole (3 micrograms/ml) was used to depolymerize microtubules in the cell. The perifusion fluid was Minimum Essential Medium containing 26.7 microCi/ml 3H-testosterone and 1.3 microCi/ml 14C-polyethyleneglycol (14C-PEG), or the same fluid supplemented with cycloheximide or nocodazole. Radioactivity of 3H-androgen and 14C-PEG in perifusion and intraluminal fluids was determined at one hour after initiation of the sustaining perifusion, and the percentage of radioactivity of 3H-androgen and 14C-PEG appearing in the intraluminal fluid to that in the peritubular fluid was determined. Proluminal movement of 3H-androgens into the caput epididymal tubules in the control rats was 323.4 +/- 73.2%. This value was significantly reduced to 121.8 +/- 13% by addition of cycloheximide to the perifusion fluid (p < 0.01). Transepithelial movement of 3H-androgen in the caput epididymis was significantly decreased to 86.6 +/- 5.3% by exposure of the epididymal tubules to nocodazole (p < 0.01). Inhibition of protein synthesis and disassembly of microtubules in the epididymal epithelial cells completely eliminated antigrade proluminal movement of 3H-androgen into the tubules. Study of the incorporation of 35S-Methionine into epididymal tissue protein revealed significant reduction of the quantity of radiolabeled proteins in the perifused tissue with fluid containing cycloheximide (p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

The Effect of Protein Synthesis Inhibition on Petal Senescence in Cut Bulb Flowers

Petal opening and senescence of cut Gladiolus, Iris, and Narcissus flowers was significantly inhibited by continuous treatment with 1 mm CHI. Vase life was doubled in individual flowers treated when half-open, and a similar effect was detected after pulsing cut gladiolus spikes with 1 mm CHI for 24 hours. Petal wilting was markedly inhibited in flowers treated with CHI and was confined to the outer 2 to 3 mm of petal margins as opposed to the entire petal in untreated flowers. These effects were not seen, however, in CHI-treated cut tulip flowers, where vase life was significantly reduced. CHI markedly inhibited protein synthesis in Gladiolus `New Rose' florets (a decrease of &gt;60%). Treatment with a potent biocide, DICA, did not increase vase life; therefore, CHI was not prolonging flower longevity by preventing microbial growth in the vase solution. The results indicate that de novo protein synthesis is required for bulb flower development and opening and petal wilting and senescence. Chemical names used: cycloheximide (CHI), sodium dichloroisocyanuric acid (DICA).

Messenger RNA decay of macrophage colony-stimulating factor in human ovarian carcinomas in vitro.

Recently, the importance of the macrophage colony-stimulating factor (CSF-1) and its receptor (encoded by the c-fms proto-oncogene) in patients with epithelial ovarian cancer has been recognized. Overexpression of CSF-1 denotes poor prognosis. Macrophage colony-stimulating factor may be one of a group of inflammatory cytokines, whose 3' untranslated region (UTR) contains AU-rich stretches and whose expression may be largely dependent on mRNA decay. The purposes of this study were to investigate the effect of protein synthesis inhibition on CSF-1 transcript expression, to help determine whether there is a role for labile intermediary proteins in the regulation of CSF-1 expression, and to explore the transcriptional or post-transcriptional mechanisms that could underlie such overexpression of CSF-1 transcripts by protein synthesis inhibitors. Although regulation of CSF-1 gene expression has been investigated in hematopoietic cells, such studies have not been carried out in any epithelial cancer. Northern blot analyses for CSF-1 expression were performed on total cellular RNA extracted from primary and established ovarian cancer cell lines in the absence or presence of proteins synthesis inhibitors with different modes of action. The probe for the AU-rich exon 10 of CSF-1 was prepared by amplification of the terminal 143 bp of the 3' UTR of human CSF-1 by polymerase chain reaction. Transcription rates were assessed in the presence or absence of cycloheximide (CH) in nuclei of ovarian cancer cells by run-off analyses. The effect of CH on CSF-1 mRNA half-life was measured by actinomycin D chase experiments in SKOV3 cells. We demonstrate that CSF-1 mRNA was expressed by all of a panel of primary and established ovarian cancer cell lines. There are at least three CSF-1 transcripts expressed by ovarian cancer cells; we demonstrate that the 4.2-kb CSF-1 transcript contains exon-6 sequences, which are spliced from the 3.4- and 1.9-kb transcripts, whereas both the 4.2- and 3.4-kb CSF-1 transcripts contain the 3' AU-rich exon 10. Treatment with several different protein synthesis inhibitors resulted in marked overexpression of CSF-1 transcript levels, suggesting a potential role for labile proteins in the regulation of CSF-1 expression. The predominant effect of CH is on the two CSF-1 transcripts that contain exon 10. Although CH does not change the rate of CSF-1 gene transcription, measurement of CSF-1 mRNA stability reveals a prolongation of CSF-1 transcript half-life by CH from 4.5 hours to significantly greater than 6 hours in ovarian cancer cells. Augmented CSF-1 transcript stability underlies the marked overexpression of CSF-1 seen with protein synthesis inhibitors. Our results suggest the involvement of a labile regulatory protein that contributes to CSF-1 mRNA decay in ovarian cancer cells. Our data suggest further that exon 10 (not the spliced exon-6 sequences) of the CSF-1 transcript may contain an instability determinant.

Post-transcriptional regulation of c- fms proto-oncogene expression by dexamethasone and of CSF-1 in human breast carcinomas in vitro

The c- fms proto-oncogene encodes the receptor for a hematopoietic growth factor, CSF-1. Recently, the importance of c- fms and its ligand CSF-1 in malignancies of non-hematopoietic origin, such as breast, ovarian, endometrial, pulmonary, and trophoblastic cancers has been recognized. We have previously shown that glucocorticoids induce a large increase in c- fms mRNA and protein levels in breast carcinoma cell lines. In this report, we investigate the mechanism underlying such c- fms overexpression by dexamethasone. We show that dexamethasone treatment of two breast carcinoma cell lines (BT20-c- fms expressor, and SKBR3-co-expressor of both c- fms and CSF-1) does not increase the rate of c- fms gene transcription, suggesting a post-transcriptional mechanism of regulation of c- fms expression by dexamethasone. The effect of protein synthesis inhibition was studied to help determine whether there was a role for intermediary regulatory proteins in the regulation of c- fms expression. We find that several protein synthesis inhibitors interfere with dexamethasone induction of c- fms transcripts, suggesting the existence of regulatory proteins. These regulatory proteins do not appear to be constitutively expressed, as we show no effect of protein synthesis inhibition on c- fms transcript expression in resting BT20 cells. These findings suggest that the putative regulatory proteins are induced by dexamethasone. Furthermore, the addition of a protein synthesis inhibitor, pactamycin, to dexamethasone-treated BT20 cells results in a decrease in c- fms mRNA stability. There is no decrease of c- fms transcripts over the six hours of actinomycin-D chase in the dexamethasone-treated cells. However, when pactamycin is added to dexamethasone, the calculated c- fms mRNA half-life falls to seven hours, which represents a decrease of c- fms mRNA half-life to that measured in the resting cell, and in monocytes. Taken together, these results suggest the involvement of a dexamethasone-inducible c- fms mRNA stabilizing protein in the regulation of c- fms gene expression in breast carcinoma cell lines. Interestingly, dexamethasone has no effect on CSF-1 expression in SKBR3 cells. Protein synthesis inhibition however results in a reciprocal effect, with induction and stabilization of CSF-1 transcripts, which contrasts with the destabilization of c- fms mRNA. This reciprocal effect led us to postulate that CSF-1 may also contribute to the post-transcriptional regulation of c- fms mRNA expression in breast carcinoma cells. Therefore, the effect of addition of exogenous CSF-1 on c- fms mRNA expression in BT20 cells was studied. We demonstrate that exogenous CSF-1 is able to down-regulate c- fms mRNA expression by 3-fold in BT20 cells. This suggests that CSF-1, in addition to glucocorticoids, may play a role in the post-transcriptional regulation of c- fms expression in breast carcinoma cells. (Steroids 59:514–522, 1994)

Transient cold shock induces the heat shock response upon recovery at 37 degrees C in human cells.

We evaluated the effects of a transient cold shock followed by recovery and incubation at 37 degrees C on the regulation of heat shock gene expression in the IMR-90 human diploid fibroblasts and HeLa cells in tissue culture. We showed that preincubation of cells at 4 degrees C induced the synthesis and accumulation of the heat shock proteins (HSPs) upon recovery at 37 degrees C, and the degree of this induction was directly related to the time that the cells spent at 4 degrees C. Assays on the abundance of the hsp 70 transcript, the hsp 70 gene promoter activity, and the trimerization and activation of heat shock factor (HSF) to bind to its consensus heat shock element (HSE) provided evidence that this induction of the heat shock response in cells recovering from a transient cold shock is attributable to a transcriptional event mediated by the activation of HSF. Further, the induction was a response to the temperature upshift from 4 to 37 degrees C as opposed to the 4 degrees C treatment itself; quantitation of the HSE-binding activity of cells incubated at 4 degrees C without recovery and incubation at 37 degrees C gave no evidence of an activated response. Analysis of the effects of protein synthesis inhibitors demonstrated that neither cycloheximide nor puromycin was effective in blocking the induction of HSE-binding activity in cells recovering from a transient cold shock. Experiments on the time course and temperature dependence of this induction of HSE-binding activity showed that the onset, magnitude, and duration of this induction were directly proportional to the severity of the cold stress (measured by time and temperature). We discuss the possible mechanism(s) involved in this induction of the heat shock genes at 37 degrees C by a transient cold shock and the biological implications of this observation.

Relationship between photoinhibition of photosynthesis, D1 protein turnover and chloroplast structure: effects of protein synthesis inhibitors

ABSTRACTIrradiation of Spinach oleracea intact leaf tissue and of mesophyll protoplasts of Valerianella locusta at 20° C with strong light resulted in severe (40–80%) inhibition of photosynthesis, measured as photosystem II electron transport activity in isolated thylakoids or as fluorescence parameter FV/FM on intact leaf disks. No net degradation of the D1 protein of photosystem II was seen under these conditions. However, in the presence of streptomycin, an inhibitor of chloroplast protein synthesis, net D1 degradation (up to about 80%) did occur with a half‐time of 4–6h, and photoinhibition was enhanced. Thylakoid ultrastructure remained stable during photoinhibition, even when substantial degradation of D1 took place in the presence of streptomycin. When leaf disks were irradiated at 2°C, streptomycin did not influence the degree of photoinhibition, and net Dl degradation did not occur. These results suggest that in excess (photoinhibitory) light at 20°C, turnover (coordinated degradation and synthesis) of D1 diminished the degree of photoinhibition. The observed photoinhibition is thought to be due to the accumulation of inactive photosystem II reaction centres still containing D1. In the presence of streptomycin, the Dl protein was degraded (probably in the previously inactivated centres), but restoration of active centres via D1 synthesis was blocked, leading to more severe photoinhibition. Low temperature (2°C), by restricting both degradation and resynthesis of D1, favoured the accumulation of inactive centres. Streptomycin and chloramphenicol (another inhibitor of chloroplast protein synthesis) were tested for side‐effects on photosynthesis. Strong inhibitory effects of chloramphenicol, but much less severe effects of streptomycin were observed.

Rapid Accumulation of Phenylalanine Ammonia-Lyase mRNA and 3-Hydroxy-3-Methylglutaryl CoA Reductase mRNA by Protein Synthesis Inhibitors

Hyphal wall components of Phytophthora infestans, the late blight fungus, induced rapid accumulation of phenylalanine ammonia-lyase (PAL) and 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) transcripts (mRNA) in potato tubers. The effects of protein synthesis inhibitors, blasticidin S and cycloheximide, on the gene transcription were examined. These inhibitors also induced PAL and HMGR transcript accumulation.

Effect of protein synthesis inhibitors on neuronal mechanisms of sensitization inHelix snail

A sensitizing treatment with 5–10% quinine solution causes short-term (lasting 50–70 min) and long-term (lasting several hours) changes in the activity of the command neurons for defensive behavior (LPl1 and PPl1) in the snailHelix lucorum. The short-term effects are characterized by a depolarizing shift in membrane potential, increased excitability, and an initial increase in the content of bound calcium (Ca-c) in the neurons. The long-term effects appear as facilitation of synaptic components of neuronal responses to sensory stimuli without any changes in excitability and in membrane potential, and also as a repeated increase of Ca-c content. Treatment with anisomycin or cycloheximide during sensitization acquirement prevents development of long-term sensitization.

Inhibition of the degradation of the precursor and of the mature β1 integrin subunit by different protein synthesis inhibitors and by ATP depletion

A stabilization of both the precursor and the mature β1 integrin subunit was observed in metabolically labeled human skin fibroblasts and Molt-4 T lymphocytes upon addition of protein synthesis inhibitors or by ATP depletion. Differentia] effects of protein synthesis inhibitors are reported since the slow degradation of the mature β1 subunit was sensitive to cycloheximide but not to puromycin. We also show that the half-life of the mature subunit was not dependent on intracellular lysosomal degradation or on ubiquitination suggesting that VLA turn-over occurs at the cell surface and might involve proteins or proteases with short half-life.

The effect of ongoing protein synthesis on the steady state levels of Gp63 RNAs in Leishmania chagasi

G63, the major surface glycoprotein of Leishmania chagasi promastigotes, increases 11-fold in amount as promastigotes grow from logarithmic to stationary phase. Transcripts from three different classes of gp63 genes are differentially expressed during this development (Ramamoorthy, R., Donelson, J. E., Paetz, K. E., Maybodi, M., Roberts, S. P., and Wilson, M. E. (1992) J. Biol. Chem. 267, 1888-1895). We studied the effect of protein synthesis inhibitors on gp63 mRNAs. The steady state level of log class gp63 RNA, expressed primarily in logarithmic phase promastigotes, increased 16.5-fold after incubation in cycloheximide. A similar increase in log gp63 RNAs was caused by inhibitors that block different steps in translation. In contrast, the levels of stationary class gp63 RNA, expressed in stationary phase parasites, and constitutive class gp63 RNA, expressed throughout promastigote growth, increased only 2.3- and 1.5-fold, respectively. The latter was not statistically significant. Nuclear run-on assays showed that the cycloheximide effect was not due to an increased rate of transcription. However, the t1/2 of log RNAs was prolonged 6.5-fold after incubation in cycloheximide, in contrast to a 1.7-fold increase in the t1/2 of ATPase RNA, suggesting that cycloheximide specifically stabilizes log gp63 mRNAs. Thus, a highly labile negative regulatory protein, such as an RNase, may specifically target log gp63 RNAs for degradation.

Inhibition of protein synthesis separates autophagic sequestration from the delivery of lysosomal enzymes

To investigate the role of newly synthesized proteins during autophagic sequestration and degradation, the effects of protein synthesis inhibition on autophagic vacuole (AV) formation and degradation were analyzed. The inhibition of protein synthesis was found to separate autophagic sequestration from the delivery of lysosomal enzymes to (AVs). Pretreatment with cycloheximide for > or = 3 h caused a drastic inhibition of autophagy-induced degradation. Surprisingly, morphological analyses showed that the inhibition of protein synthesis for up to 12 h did not block the formation of nascent AVs; however, it did prevent their conversion into degradative AVs. Using immunoperoxidase cytochemistry with an antibody against cathepsin D and labeling of lysosomes with endocytosed colloidal gold, we found that the nascent AVs that formed during prolonged cycloheximide pretreatment had not received lysosomal markers. The inhibition of autophagic degradation and lysosomal enzyme delivery were rapidly reversed following the removal of cycloheximide. These results suggest that there is a fairly rapid turnover of protein(s) that are necessary for lysosomal fusion, but that the initial formation of AVs is independent of new protein synthesis for a long period of time.

Induction of Tyrosine Hydroxylase mRNA by Protein Synthesis Inhibitors in the Rat Adrenal

To facilitate the quantitation of the steady-state levels of tyrosine hydroxylase (TH) mRNA, we have developed a specific, rapid, and sensitive solution hybridization assay. By use of this assay, the induction of TH mRNA by protein synthesis inhibitors (PSIs) in the rat adrenal was investigated. Treatment with PSIs, either cycloheximide (CHX) or anisomycin (ANI), increased the levels of TH mRNA in the adrenal. The levels of adrenal TH mRNA were increased 1 h after CHX and had increased by approximately threefold over saline control [from 3.85 ± 0.4 (SE) to 11.5 ± 2 (SE) pg/μg RNA] at 6 h after treatment. A similar time course was observed after ANI treatment. These in vivo kinetics cannot be accounted for solely by the stabilization of TH mRNA. Northern blot analysis indicated that the size of the TH mRNA in the adrenal was not altered by PSIs. The CHX induction of adrenal TH mRNA was dose-dependent in the range of 2 to 4 mg/kg, sc, and was not further increased by a dose of 50 mg/kg, sc. Unilateral adrenal denervation did not alter the CHX induction of TH mRNA in medullae. These results suggest that PSIs exert a direct effect on the adrenal medulla. These effects of PSIs may be mediated by a labile repressor which regulates adrenal TH gene expression in vivo.

Epidermal growth factor induces prolactin mRNA in GH 4C 1 cells via a protein synthesis-dependent pathway

Prolactin (PRL) gene expression is regulated through a complex network of signal transduction pathways activated by various hormones and growth factors. Estrogens regulate PRL gene transcription in vivo through both direct and indirect, protein synthesis-dependent, mechanisms. Therefore, we hypothesized that other stimulators of PRL gene transcription might also act via protein synthesis-dependent mechanisms. To test this hypothesis, we examined, in GH 4C 1rat pituitary tumor cells, the effects of protein synthesis inhibitors on the induction of PRL mRNA by known stimulators of PRL gene transcription. Whereas induction by epidermal growth factor (EGF) was abolished by cycloheximide and puromycin, increases in PRL mRNA caused by thyrotropin releasing hormone, 12- O-tetradecanoylphorbol 13-acetate, forskolin, or dibutyryl cyclic AMP were unaffected. These data suggest that the induction of PRL mRNA by EGF may require the induced synthesis of an intermediary regulatory protein.

Antibodies against Fungal Conidia and Antibiotics Inhibit Phenylalanine Ammonia-Lyase Activation in Citrus

Summary Antibodies raised against Trichoderma harzianum conidia prevented accumulation of Phenylalanine ammonia-lyase (PAL, E.C. 4.3.1.5.) in Citrus . The observed effect of the antibodies is produced by a delay in the formation of the fungal germination tube. An increase of PAL activity of ca. 7 fold in C. limon and C. sinensis seedlings, and of ca. 2 fold in C. paradisii seedlings was found, as an hypersensitive response to inoculation with T. harzianum conidia. This increase in activity was due to «de novo« synthesis of the enzyme as demonstrated by the effect of protein-synthesis inhibitors and by immunotitration.

Relaxin secretion by porcine large luteal cells: effect of protein synthesis inhibitors.

The purpose of the experiments reported herein was to investigate the relative importance of new hormone synthesis to basal and prostaglandin E2-stimulated rates of relaxin release. A relaxin-reverse hemolytic plaque assay was used to monitor relaxin release from individual large luteal cells (LLC) in which new protein synthesis was inhibited by cycloheximide or actinomycin D. These treatments significantly decreased the rate of relaxin release. In addition, cycloheximide reduced the total fraction of LLC possessing the ability to form plaques by about 10%, suggesting complete suppression of relaxin from this subset of cells. Exposure of inhibitor-treated LLC to prostaglandin E2 (a relaxin stimulatory secretagogue) enhanced relaxin release, and restored suppressed LLC back into the secretory population. Taken overall, these results demonstrate that the majority of relaxin-releasing LLC exploit a mixture of newly synthesized and older, stored hormone to achieve basal secretion. A minority of relaxin-releasing LLC, however, appear to depend wholly on newly synthesized hormone for basal secretion. The differential activity (and interaction) of these pathways in individual LLC may provide a potential explanation for the markedly heterogenous manner of hormone release observed in this (and other) cell types, and for the action of relaxin secretagogues.

Activation by Protein Synthesis Inhibitors of Glucose Transport into L6 Muscle Cells

The effects of protein synthesis inhibitors on glucose transport into L6 myotubes was examined. Similar to the acute effects of insulin, short-term treatment with anisomycin or cycloheximide increased transport 2-fold. Kinetic studies demonstrated that this activation was due to a doubling in the V max values. The effects of anisomycin or cycloheximide were not additive to that of insulin. Both protein synthesis inhibitors caused only small increases in GLUT4 expression and negligible effects on GLUT1 expression. Anisomycin, as well as insulin, induced the translocation of both transporters from intracellular vesicles to the plasma membrane though not in quantities sufficient to entirely account for the activation of transport. The results indicate that protein synthesis inhibitors stimulate hexose transport in L6 myotubes by increasing the number of transporters in the plasma membrane and augmenting the intrinsic catalytic activity of the transporters.

Accelerated poly(A) loss and mRNA stabilization are independent effects of protein synthesis inhibition on alpha-tubulin mRNA in Chlamydomonas.

In Chlamydomonas, the usual rapid degradation of tubulin mRNAs induced by flagellar amputation is prevented by inhibition of protein synthesis with cycloheximide. Evidence is presented that the ability of cycloheximide to stabilize alpha-tubulin mRNA depends on the time of addition. Addition of cycloheximide to cells before induction strongly stabilizes the induced mRNAs, while addition after their synthesis stabilizes them only transiently. Moreover, cycloheximide inhibition does not stabilize the same alpha-tubulin mRNA species in uninduced cells. These results suggest that cycloheximide is not acting to stabilize the induced alpha-tubulin mRNAs simply by preventing ribosome translocation. The stabilized state of tubulin mRNA was found to correlate with its occurrence on smaller polysomes but larger EDTA-released mRNP particles than the unstable state. A second effect of cycloheximide on the metabolism of induced tubulin mRNAs is to accelerate complete poly(A) removal. This effect of cycloheximide inhibition, unlike stabilization, occurs whenever cycloheximide is added to cells, and appears unrelated to stabilization. The effect is shown to be mRNA-specific; poly(A)-shortening on the rbcS2 mRNA is not altered in the presence of cycloheximide, nor do completely deadenylated molecules accumulate. Experiments in which cells were released from cycloheximide inhibition suggest that deadenylated alpha-tubulin mRNAs may be less stable than their polyadenylated counterparts during active translation.

Effect of Protein Synthesis Inhibitors on Tobacco Seed Germination and Seedling Emergence

Summary This study is based on two earlier reports, one demonstrating that dark preincubation improves tobacco (Nicotiana tabacum L.) seed germination rate and the other demonstrating genetic variability in tobacco seed response to germination temperature. Each of these situations involves special classes of proteins: the phytochrome molecule and heat shock proteins, respectively. A better understanding of either process at the molecular level would involve investigations on the regulatory role of the protein synthesis mechanism. This study was undertaken to characterize the temporal relationship of the three major steps in protein synthesis - replication, transcription and translation - with the three major events associated with seedling morphogenesis: radicle protrusion, radicle elongation, and plumule protrusion. This was accomplished through the use of selected inhibitors of replication, transcription, and translation. Results suggest that a) radicle emergence is regulated by translation but is independent of replication and transcription; b) the translational regulation of the above event is mediated through preformed (i.e., longlived) m-RNAs; c) radicle elongation following its emergence and plumule emergence are regulated by all three components of protein synthesis; d) radicle length data obtained in laboratory investigations are a better predictor than radicle number (i.e., germination percentage) of seedling emergence potential under field conditions.